Convert hifi_reads.bam to fastq for CellRanger

Question

[nwerry]

Hi all,

I've been attempting to process MAS-Seq scRNA Seq data for variant calling using souporcell and am stuck in the pre-processing stage. My starting data is a MAS-Seq hifi reads bam (m84066_231220_211345_s4.hifi_reads.bam), which I have started processing through the isoseq pipeline for adapter removal, etc.

For the final analysis in souporcell it needs the possorted_bam output from CellRanger, and I'm not sure how to generate a comparable file to this, or convert what I have to a fastq to run cellranger directly.

I've tried using smrtlink bam2fastq off the Skera-split file, but running cellranger count off that gives the chemistry error below and the cellranger bam2fastq fails due to the 10x BAM file not being recognized.

Any advice on how to proceed here?

Chemistry error:

[error] Pipestance failed. Error log at:
GFPs/SC_RNA_COUNTER_CS/SC_MULTI_CORE/MULTI_CHEMISTRY_DETECTOR/_GEM_WELL_CHEMISTRY_DETECTOR/DETECT_COUNT_CHEMISTRY/fork0/chnk0-ue839a14419/_errors

Log message:
The read lengths are incompatible with all the chemistries for Sample GFPs in "/share/vanelab/nick/NANOS3_scRNA/GFP_fastq".
 - read1 median length = 799
 - read2 median length = 0
 - index1 median length = 0

The minimum read length for different chemistries are:
SFRP     - read1: 26, read2: 30, index1: 0
SC5P-R2  - read1: 26, read2: 25, index1: 0
SC5P-PE  - read1: 81, read2: 25, index1: 0
SC3Pv1   - read1: 25, read2: 10, index1: 14
SC3Pv2   - read1: 26, read2: 25, index1: 0
SC3Pv3   - read1: 26, read2: 25, index1: 0
SC3Pv3LT - read1: 26, read2: 25, index1: 0
SC3Pv3HT - read1: 26, read2: 25, index1: 0

We expect that at least 50% of the reads exceed the minimum length.

Answer 1

[ATpoint]

CellRanger is specific to 10x Chromium scRNA-seq. Forcing other types of scRNA-seq into it will almost certainly not work. Consider using STARsolo for more flexibility if that applies here.

[nwerry]

Thanks for the response, I'm I was provided these data without a lot of context so I could be wrong here, but my understanding is the samples were prepped with the MAS-Seq for 10x Single Cell 3 kit, which uses 10x Chromium sc cDNA.

That would still not be compatible?

Thanks again, Nick